Spatial and temporal genetic heterogeneity of epidermal growth factor receptor gene status in a patient with non-small cell lung cancer: a case report.

Abstract

To date, an epidermal growth factor receptor-activating mutation is recognized as a genetic hallmark that predicts a good response to treatment with epidermal growth factor receptor tyrosine kinase inhibitor. However, there has been less long-term observation of the mutational status within the same patient. To the best of our knowledge, this is the first case report which illustrates the instability of the genetic status of pulmonary adenocarcinoma cells.
A 64-year-old Japanese woman with advanced lung adenocarcinoma had been undergoing various anticancer treatments, including epidermal growth factor receptor tyrosine kinase inhibitor, for seven years. She had been receiving locoregional treatment in addition to systemic treatment. She maintained a good performance status until seven years after the initial diagnosis, although she had local and distant recurrences. We analyzed the genetic status of the epidermal growth factor receptor gene in a series of specimens obtained from various tumor-containing lesions throughout the therapeutic period. The results of the genetic analyses clearly showed that the spatial and temporal genetic heterogeneity of the epidermal growth factor receptor gene status originated from an identical tumor ancestor.
An alternative paradigm to determine a therapeutic strategy for a patient with lung cancer should be considered given the genetic heterogeneity and instability of tumor cells.

Full text

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CASE REPORT Open Access
Spatial and temporal genetic heterogeneity of
epidermal growth factor receptor gene status in
a patient with non-small cell lung cancer: a case
report
Makoto Ogata, Toshiki Shimizu*, Takashi Yokoi and Shosaku Nomura
Abstract
Introduction: To date, an epidermal growth factor receptor-activating mutation is recognized as a genetic
hallmark that predicts a good response to treatment with epidermal growth factor receptor tyrosine kinase
inhibitor. However, there has been less long-term observation of the mutational status within the same patient. To
the best of our knowledge, this is the first case report which illustrates the instability of the genetic status of
pulmonary adenocarcinoma cells.
Case presentation: A 64-year-old Japanese woman with advanced lung adenocarcinoma had been undergoing
various anticancer treatments, including epidermal growth factor receptor tyrosine kinase inhibitor, for seven years.
She had been receiving locoregional treatment in addition to systemic treatment. She maintained a good
performance status until seven years after the initial diagnosis, although she had local and distant recurrences. We
analyzed the genetic status of the epidermal growth factor receptor gene in a series of specimens obtained from
various tumor-containing lesions throughout the therapeutic period. The results of the genetic analyses clearly
showed that the spatial and temporal genetic heterogeneity of the epidermal growth factor receptor gene status
originated from an identical tumor ancestor.
Conclusions: An alternative paradigm to determine a therapeutic strategy for a patient with lung cancer should
be considered given the genetic heterogeneity and instability of tumor cells.
Background
Epidermal growth factor receptor (EGFR) tyrosine
kinase inhibition is an active strategy in non-small cell
lung cancer (NSCLC) [1]. The response to EGFR tyro-
sine kinase inhibitor (EGFR-TKI) has been shown to be
closely related to the somatic activating mutation of the
EGFR gene in tumor cells [2]. EGFR mutation has been
recognized as a crucial step in the transformation of
alveolar epithelial cells. A recent report also suggested
that the activating mutation of the EGFR gene occurs as
an early event during carcinogenesis of lung cancer [3].
Discordance in the mutation status of the EGFR gene in
the primary tumor and corresponding metastatic tumor
is occasionally observed [4-6]. However, there has been
less long-term observation of the mutational status of
the EGFR gene in the same patient. We report a series
of analyses of the EGFR gene status of a patient. The
results of our analyses clearly demonstrate a spatial and
temporal genetic heterogeneity, including double-acti-
vating mutation, in this patient.
Case presentation
A 64-year-old Japanese woman was admitted to our
hospital seven years ago with a complaint of pain in her
right hip joint. Radiographic analysis revealed an osteo-
lytic tumor of her right pelvis and a tumor in her right
lower lung field. The histological findings of a biopsy
specimen obtained from the bone and pulmonary
tumors showed adenocarcinoma. Immunohistochemical
tests showed that the tumor cells stained positive for
* Correspondence: shimizto@takii.kmu.ac.jp
First Department of Internal Medicine, Kansai Medical University, 10-15
Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan
Ogata et al. Journal of Medical Case Reports 2011, 5:553
http://www.jmedicalcasereports.com/content/5/1/553
JOURNAL OF MEDICAL
CASE REPORTS
© 2011 Ogata et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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thyroid transcription factor-1. Therefore, we diagnosed
our patient with advanced lung cancer (cT2N2M1). She
received systemic chemotherapy with carboplatin and
paclitaxel, starting one month after diagnosis after pal-
liative irradiation of the pelvic lesion. After completion
of four consecutive courses of chemotherapy, a partial
response was achieved. However, local recurrence
occurred six months later. Because docetaxel, gemcita-
bine and vinorelbine were all insufficient for inhibiting
disease progression, gefitinib was administered as the
fourth regimen, starting one year after diagnosis. A
tumor response was subsequently observed and the
treatment was continued. However, a routine brain mag-
netic resonance imaging scan showed a de novo meta-
static lesion in her left frontal lobe two years after
diagnosis. In accordance with our patient’s wishes, gefiti-
nib administration was continued after surgical resection
of the brain tumor. Although the primary lesion did not
exhibit regrowth, additional brain and pulmonary metas-
tases in her right lung were observed four years after
diagnosis. Erlotinib was administered as the fifth regi-
men following stereotactic radiosurgery for the brain
tumor. Significant growth of the pulmonary metastatic
lesion was observed one year later, although the other
lesions did not demonstrate regrowth. We repeated a
bronchoscopy for the pulmonary metastatic lesion to
investigate the EGFR gene mutation status.
We used a combination of the peptide nucleic acid-
locked nucleic acid polymerase chain reaction (PNA-
LNA PCR) clamp method and the direct sequencing
method for determining the EGFR gene mutation status
[7]. The result of the PNA-LNA PCR clamp assay for
the EGFR gene showed a double-activating mutation
consisting of an in-frame deletion mutation in exon 19
and an L858R point mutation in exon 21. The mutation
identified in exon 19 was consistent with I744-R748del
and two subsequent substitution mutations, E749I
(GAA to ATT) and A750K (GCA to AAA). To shed
light on the sequential changes in the EGFR mutation
status, we also analyzed a series of paraffin-embedded
samples obtained from this patient’s tumors. The histo-
logical findings of the analyzed samples clearly demon-
strated the presence of adenocarcinoma cells (Figure 1).
The results are summarized in Table 1. Genetic analysis
of the specimen from the primary pulmonary tumor at
diagnosis showed a wild-type EGFR gene. The specimen
Figure 1 Histological findings for the various tumor specimens, with hematoxylin and eosin staining clearly demonstrating the
presence of adenocarcinoma cells. All the original magnifications are ×200; (A) bone metastasis, (B) primary pulmonary tumor; (C) brain
metastasis; (D) pulmonary metastasis.
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from the metastatic bone tumor had an exon 19 dele-
tion identical to that in the metastatic pulmonary
tumor. L858R was not observed in the bone tumor. The
resected brain tumor harbored T790M in addition to
the exon 19 deletion. In contrast, T790M was not found
in the specimen from the pulmonary metastatic tumor.
These findings strongly suggest genetic instability and
heterogeneity of the lung tumor in this case.
We used oral TS-1 as the next regimen and achieved a
good tumor response. However, she relapsed again. She
received a salvage chemotherapy regimen comprising car-
boplatin and pemetrexed with bevacizumab, but this failed
to inhibit tumor progression. We re-biopsied the pulmon-
ary nodule by using computed tomography-guided needle
biopsy. Gene analysis of the re-biopsy specimen revealed a
unique deletion mutation in exon 19 and T790M. The
L858R mutation was not found in this specimen. Subse-
quently, our patient received thoracic irradiation for the
pulmonary nodule for locoregional control.
Discussion
An EGFR double-activating mutation involving an exon
19 deletion and L858R was first reported by Sriuran-
pong et al. [8]. Subsequently, in a study involving 145
NSCLC patients, five cases of EGFR double-activating
mutation were reported by Zhang et al. [9]. They also
showed that the exon 19 deletion and L858R were
located on the same allele. A double-activating mutation
was also reported in Japan, found in four of 111 samples
by Masago et al. [10]. Hata et al. analyzed complex
mutations in the EGFR gene in 783 NSCLC patients
[11]. They found 21 double mutations. The prevalence
of the double-activating mutation was 0.3% to 3.6%. The
biological role of the double-activating mutation with
regards sensitivity to EGFR-TKI remains controversial.
The genetic background of tumor cells in lung cancer
is not homogenous. Taniguchi et al. showed that
NSCLC cells contain a heterogeneous population of
both mutated and non-mutated EGFR tumor cells [12].
Jiang et al. also demonstrated the intratumor
heterogeneity of EGFR mutations by using a microdis-
section-based method [13]. In addition, three indepen-
dent studies also showed discordance in the EGFR gene
status of the primary tumor and corresponding meta-
static tumor. Kalikaki et al. showed that the EGFR
mutation status differed between primary tumors and
corresponding metastases in seven (28%) of 25 patients
[4]. The other two studies reported identical results
[5,6]. These findings clearly show the genetic heteroge-
neity of lung cancer.
There is increasing interest in how such genetic het-
erogeneity can be generated. The multicentric carcino-
genesishypothesis cannot
phenomenon. In the case of our patient, five indepen-
dent EGFR clones, including the wild type, were identi-
fied. The four mutant clones had a unique consensus
deletion mutation in exon 19 accompanied by two
amino acid substitutions. If mutation occurs at random,
the probability of an identical mutation occurring twice
in the same patient would be nearly equal to the univer-
sal probability bound. Thus, all four tumor clones were
from blood relatives with an identical genetic ancestor.
In terms of the cancer stem cell hypothesis, cancers
arise from stem cells that have accumulated oncogenic
mutations for transformation [14]. The transformed
stem cell transforms into the cancer stem cell, which is
capable of self-renewal and differentiation. A tumor
hierarchy consisting of a small number of quiescent can-
cer stem/progenitor cells and a large number of prolifer-
ating effector tumor cells may be generated. However,
only a cancer stem cell can reproduce another cancer
stem cell. Therefore, the genetic information found in
tumors may be inherited from cancer stem cells. Spora-
dic and step-by-step accumulations of mutations in can-
cer stem cells can generate a variety of tumor clones
with a distinct genetic background.
The sequential accumulation of de novo mutations in
tumor cells makes it difficult to predict sensitivity to
EGFR-TKI. It is possible that a tumor with wild-type
EGFR before front-line chemotherapy can become an
alternative tumor, harboring the EGFR-activating muta-
tion. An accumulation of alternative mutations, including
a T790M-insensitive mutation, in addition to the primary
activating mutation could modulate the sensitivity to
EGFR-TKI. Therefore, if available, frequent re-analyses of
the EGFR gene status may be required during a long-
term clinical course. Repeated biopsies often inflict pain
on patients, especially those with NSCLC. Thus, physi-
cians must carefully determine the need for additional
sampling based on the risk and benefit to the patient.
fully explain this
Conclusion
Spatial and temporal observation of the EGFR gene sta-
tus could help in making an appropriate therapeutic
Table 1 The genetic heterogeneity of EGFR gene status
in our patient.
Sampling TimeOrigin Exon 19
deletiona
L858RT790M
At initial diagnosis Bone
Lungb
PositiveNegative Negative
At initial diagnosis
Negative Negative Negative
Three years after
diagnosis
BrainPositiveNegative Positive
Five years after
diagnosis
Six years after diagnosis Lungc
Lungc
PositivePositiveNegative
PositiveNegative Positive
aI744-R748del, E749I (GAA to ATT), and A750K (GCA to AAA).bPrimary tumor.
cPulmonary metastatic nodule.
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decision for locoregional and systemic management of
lung cancer. An alternative paradigm to determine a
therapeutic strategy for a patient with lung cancer war-
rants consideration based on the genetic heterogeneity
and instability of the tumor cells.
Consent
Written informed consent was obtained from the patient
for publication of this case report and any accompany-
ing images. A copy of the written consent is available
for review by the Editor-in-Chief of this journal.
Abbreviations
EGFR: epidermal growth factor receptor; EGFR-TKI: epidermal growth factor
receptor tyrosine kinase inhibitor; NSCLC: non-small cell lung cancer; PNA-
LNA PCR: peptide nucleic acid-locked nucleic acid polymerase chain
reaction.
Authors’ contributions
MO prepared the molecular genetic analyses and drafted the manuscript. TY
and SN participated in drafting the manuscript. TS carried out the patient’s
therapy and helped to draft the manuscript. All authors read and approved
the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 3 June 2011 Accepted: 22 November 2011
Published: 22 November 2011
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doi:10.1186/1752-1947-5-553
Cite this article as: Ogata et al.: Spatial and temporal genetic
heterogeneity of epidermal growth factor receptor gene status in a
patient with non-small cell lung cancer: a case report. Journal of Medical
Case Reports 2011 5:553.
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